Bone marrow is a complex tissue. Principally, marrow is the site for the differentiation and maturation of hematopoietic cells. From pluripotent stem cells, a number of cell lineages differentiate. The primary components among these lineages include: the lymphoid (B, T and NK cells), the erythroid (red blood cells) and the myeloid lines (basophils, neutrophils, eosinophils, megakaryocytes and macrophages). At any given time, each of these lineages typically is present in a marrow sample.
Once differentiated, each of the various lineages matures through several stages. Maturation occurs in the marrow and elsewhere. For example, in the erythroid line, the red blood cell begins in marrow as a blast cell having a defined morphology. From that stage to the reticulocyte stage, a number of additional morphological changes occur throughout a number of stages culminating with the extrusion of the nucleus to form the reticulocyte. The reticulocyte then is released to the blood where it undergoes its final maturational change to become an erythrocyte. Other lineages undergo similar development, although the number of stages of development and the location may differ (e.g. immature T cells begin to develop in marrow but the primary location for maturation and differentiation is in the thymus). By examining a sample of bone marrow, therefore, one can identify not only the various lineages within the sample but also the several stages within each lineage.
It is important to be able to identify the lineages and/or developmental stages in a sample. The results may provide an indication of a clinical condition. For example, in a patient with anemia, in order to treat the cause of the condition one must know whether it is the result of a destruction of red cells in the blood or the result of a failure of the blast cells to mature. By examining the marrow, the presence (or absence) of stem cells, blasts and reticulocytes in normal amounts can rule out (or confirm) a diagnosis. Thus, by distinguishing a change from the "normal" condition, abnormal events can be diagnosed.
To identify the various lineages and developmental stages, one must examine a sample in great detail. It is known that each lineage and each stage within a lineage has certain defined morphological characteristics. Thus, the current practice is to take a sample of marrow by either aspiration or biopsy, stain it and examine it under a light microscope. Such a procedure is subjective, time-intensive, semi-quantitative and may require multiple stainings in order to obtain gross results.
In addition to gross morphological changes that occur with maturation and differentiation, other detailed changes occur in gene expression. A number of studies have described the appearance (or disappearance) during maturation of a variety of such cell surface antigens in a single lineage (e.g., normal erythrocytes) and for a single antigen expressed on several lineages during their maturational process. See, e.g., Loken et al., Flow Cytometric Analysis of Human Bone Marrow: I. Normal Erythroid Development, Blood, 69:255-263 (1987), and see Civin et al., Antigenic Analysis of Hematopoiesis. VI. Flow Cytometric Characterization of MY-10-positive Progenitor Cells in Normal Human Bone Marrow, Exp. Hematol., 15:10-17 (1987) respectively. Because of the cellular heterogeneity of cells within marrow, the use of markers to detect cell surface antigens was not fully sufficient to distinguish between lineages.
Thus, there was no known means to identify and distinguish between lineages and stages of development in single procedure. Such a method would provide a unique and rapid technique for the diagnosis of abnormal hematopoietic conditions.